Received: 8 April 2011 Accepted: 15 September 2011 Published online: 14 October 2011

Abstract

Skeletal
muscle innervation is a multi-step process leading to the neuromuscular
junction (NMJ) apparatus formation. The transmission
of the signal from nerve to muscle occurs at the NMJ level.
The molecular mechanism that orchestrates the organization and
functioning of synapses is highly complex, and it has not
been completely elucidated so far. Neuromuscular junctions are assembled
on the muscle fibers at very precise locations called end
plates (EP). Acetylcholine receptor (AChR) clusterization at the
end plates is required for an accurate synaptic
transmission. This review will focus on some mechanisms responsible for
accomplishing
the correct distribution of AChRs at the synapses. Recent
evidences support the concept that a dual transcriptional control
of AChR genes in subsynaptic and extrasynaptic nuclei is
crucial for AChR clusterization. Moreover, new players have been
discovered in the agrin–MuSK pathway, the master organizer
of postsynaptical differentiation. Mutations in this pathway cause
neuromuscular congenital disorders. Alterations of the
postynaptic apparatus are also present in physiological conditions
characterized by skeletal muscle wasting. Indeed, recent
evidences demonstrate how NMJ misfunctioning has a crucial role at
the onset of age-associated sarcopenia.

Motor neuron innervation is the most
relevant maturation event in skeletal muscle development since muscle
physiology, also
accounting for locomotion and breath, depends on it.
Innervation implies sophisticated interactions between motor neuron
axons
and end plates on the muscle fibers, thus resulting in a
development of highly elaborated synapses called neuromuscular junctions
(NMJs). As soon as the action potential reaches the axon
terminals, it induces the opening of the voltage-gated Ca2+ channels on the presynaptic nerve membrane. This allows a Ca2+
influx that induces synaptic vescicles to fuse with the presynaptic
membrane and to release the neurotransmitter acetylcholine
(ACh) in the synaptic cleft. From here, ACh diffuses and
binds to acetylcoline receptors (AChRs) localized on the postsynaptic
membrane on the muscle fiber. This binding makes AChRs
permeable to both Na+ and K+ and opens the associated voltage-gated Na+ channels on the muscle membrane which, in turn, initiate an action potential causing Ca2+
release from the sarcoplasmic reticulum into the cytosol and muscle
contraction. Acetylcholinesterase, located on the synaptic
portion of the basal lamina that envelops muscle fibers,
quickly inactivates ACh released from the presynaptic membrane so
that the ACh concentration in the synaptic cleft
decreases rapidly and neurotransmission stops. The correct nerve–muscle
impulse
transmission requires an intricate network of interacting
signaling pathways displaying a certain rate of redundancy, which
is, however, necessary to ensure the process’s spatial
and temporal accuracy.

Defects in the signaling pathways that
regulate NMJ differentiation and functioning lead to a variety of
congenital neuromuscular
disorders termed congenital myasthenic syndromes (CMS),
typified by muscle weakness and fatigue [1].
Loss of the nerve signaling caused by the degeneration of motor neurons
leads to a debilitating loss of muscle mass, atrophy,
and paralysis. Also, aging and diseases such as cancer,
AIDS, and chronic heart failure are physical conditions in which NMJ
normal activity, muscle mass, and function are highly
compromised. For these reasons, understanding the molecular basis of
neuromuscular synapse functioning is fundamental to such
disorders’ therapeutic approach. This review will focus on the
development
of the postsynaptic apparatus and will highlight the role
of some defects of this process which are at the onset of neuromuscular
disease and muscle wasting-associated pathologies.

2 NMJ formation

NMJs do not develop at random locations in
muscles; rather, they are assembled in a narrow central region of the
muscle fiber,
so that many NMJs are located in a row, forming an end
plate across the fiber. Each NMJ consists of an area of apposition
between a motoneuron axon branch and a single
multinucleated muscle fiber. AChRs localize on the postsynaptic muscle
membrane,
and their spatial distribution is critical for synaptic
function. A high density of AChRs at synapses is required to initiate
a synaptic action potential in the myofiber. Vice versa,
around synapses and in the rest of the fiber, the density of AChRs
has to be kept low in order to allow a complete
maturation of the NMJ. When myoblasts fuse to form myotubes, AChR
subunits
are assembled into the membrane at a very low density
(1,000 μm−2). In mature synapses, AChRs accumulate at a density >10,000 μm−2directly beneath the motor nerve terminal while they drop to <10 μm−2 in the extrasynaptic membrane [2, 3].

A puzzling question about the developing
postsynaptic structure is whether the motoneurons or the muscle fibers
determine
where and how the NMJs are formed. The motor nerve might
initiate AChR cluster formation (neurocentric model) or clusters
might form aneurally and then recognized by the nerve
(myocentric model) [4]. Some studies
indicate that the primitive AChR clusterization is nerve independent,
while the nerve induces only maturation
and enlargement of some of the primitive aneural AChR
clusters. During early mouse embryogenesis, between embryonic
day 12.5
(E12.5) and E14.5, aneural muscle fibers begin
spontaneously to accumulate AChRs in a central region where innervation
will
occur (prepatterning) [5–9]. This phenomenon also occurs in mutant mice where motor axons fail to contact skeletal muscle [10].
Following such nerve-independent AChR clustering, nerve terminals
overlap some prepatterned AChR clusters and, at E18.5,
the innervated clusters are enlarged, whereas the other
primitive clusters disappear in synaptic and extrasynaptic regions
[8, 9]. Conversely, other studies in cultured myotubes have indicated that motor neurons induce postsynaptic differentiation ignoring
preexisting aneural clusters [11, 12]. It has also been recently hypothesized that some already formed AChR clusters are recognized by the nerve on muscle fibers,
while others are induced by motor axons [13]. It is also possible that the myogenic and the neurogenic component role might depend on the species and on the developmental
context, although the topic keeps being highly controversial [14].

3 NMJ maturation and AChR clusterization

The mechanisms that lead to the
clusterization of ACh receptors within the postsynaptic membrane require
a precise interaction
of signals among motoneurons and skeletal muscle fibers
and occur through different modalities. It has been described that
AChRs move from the extrasynaptic region and are trapped
to the synaptic pool through a rapid lateral diffusion [2].
Clustering also induces a higher stability of the AChRs; at early
stages of development, the half-life of junctional non-clustered
AChRs is about 1 day, whereas in adult, is increased
to 8–14 days [15]. Moreover,
transcriptional regulation has a pivotal role in AChR clusterization.
Nuclei that are associated at the postsynaptic
membrane (synaptic nuclei) actively transcribe the AChR
subunit genes and other postsynaptic component genes at a higher rate
than the non-synaptic nuclei. This leads to a localized
synthesis and accumulation of AChRs [16–19].
The formation of AChR clusters depends not only on positive signals
that enhance AChR concentration in the synaptic area
but also on negative signals necessary to decrease AChR
concentration outside the synaptic area, along the entire fiber.

3.1 Positive signaling

The best characterized signaling
responsible for AChR accumulation at the NMJs is the agrin/lipoprotein
receptor-related protein
4 (Lrp4)/muscle-specific tyrosine kinase receptor
(MuSK) pathway. Agrin is a large heparan sulfate proteoglycan first
isolated
from the Torpedo electric organ [20] synthesized in the motoneurons, transported along axons, and released in the synaptic basal lamina, which surrounds the
muscle fiber [21]. Agrin is necessary for clustering AChRs at synaptic sites (Fig. 1a). Agrin is sufficient to induce AChR ectopic clusters in adult muscle and postsynaptic specialization in denervated fibers
[22, 23]. Although it is not essential for prepatterning, in agrin−/−mice, NMJs and AChRs are uniformly distributed in the muscle fiber and not clustered [8, 9, 24], and postsynaptic differentiation is inhibited.

Fig. 1 The agrin–MuSK–Lrp4 and neuregulin–ErbB pathways induce NMJ assembly positive signals. a
Agrin is released by the motor axon terminal and induces AChR
clustering, phosphorylation, and stabilization at the postsynaptic
membrane. Lrp4 associates with MuSK in the
absence of agrin. Agrin binds to the preformed MuSK–Lrp4 complex by
interacting
with Lrp4 and promotes MuSK transphosphorylation
and activation. Once phosphorylated, MuSK recruits the adapter protein
Dok-7
which binds Crk and CrkL and stimulates further
MuSK phosphorylation and kinase activity. This induces phosphorylation
and
stabilization of nascent AChR clusters. Rapsyn
is a coeffector in AChR assembly which anchorates AChRs at the muscle
membrane.
b Neuregulin (NGR-1) is released by the
nerve and induces AChR transcription in synaptic nuclei. NRG-1 acts by
binding tyrosine
kinases receptors ErbBs. ErbB phosphorylation
induced by NRG stimulates ERK and JNK kinase activity which
phosphorylates GABP-α
and GABP-β transcription factors. GABP-α
heterodimerizes with GABP-β and binds DNA at the N-box thereby enhancing
transcription
of AChR genes

Agrin acts by activating another
important player in the NMJ assembly pathway, MuSK. MuSK is a tyrosine
kinase receptor expressed
in the postsynaptic membrane of NMJs [25] where it co-localizes with AChRs [26]. The main role of agrin is to initiate MuSK autophosphorylation and activation [27] (Fig. 1a). However, MuSK can also be activated in an agrin-independent manner [28]. Indeed, in MuSK−/−fibers, no NMJs are formed, and also, prepatterning before innervation is absent [8, 9, 29]. Like AChR, MuSK gene is a target for the transcription factor myogenin (see below). Myogenin mediates MuSK upregulation
during muscle fiber denervation and MuSK downregulation upon innervation [30]. However, upon innervation, in the synaptic nuclei, MuSK is upregulated by different transcriptional pathways.

Although agrin stimulates MuSK phosphorylation, the two proteins do not interact directly, but additional proteins (coreceptors)
are engaged to allow agrin-mediated signaling in myotubes [31, 32]. A recently discovered agrin coreceptor is the transmembrane protein Lrp4 [33].
Lrp4 is concentrated at the NMJs and is necessary for agrin-stimulated
MuSK phosphorylation and AChR clustering. According
to this model, Lrp4 self-associates and interacts with
MuSK in the absence of agrin. When agrin binds to this preformed
complex,
it triggers a reorganization of MuSK, so promoting its
transphophorylation and kinase activity. Once phopshorylated, MuSK
activates signaling pathways that lead to synaptic
differentiation including clustering of AChRs [34, 35] (Fig. 1a).
Agrin activation of MuSK also leads to concentration at synapses of
other proteins such as acetylcholinesterase (AChE),
rapsyn, and neuregulin (NRG) receptors (ErbBs; see
below). Moreover, Lrp4 has a crucial role in muscle prepatterning since
it promotes MuSK phosphorylation and activation in the
absence of agrin [36].

A coeffector in AChR assembly is
rapsyn, a membrane protein associated with the cytoplasmic side of the
plasma membrane (Fig. 1a). Rapsyn anchorates AChRs at the muscle membrane from the earliest stages of synaptogenesis, including prepatterning [41]. Rapsyn also interacts with cytoskeletal proteins, thus being essential for AChR clustering. In mice lacking rapsyn, AChRs
fail to aggregate [42].
It has been proposed that MuSK induces phosphorylation of nascent AChR
clusters and promotes also the binding of additional
rapsyn to AChR aggregates, which contributes to AChR
cluster growth and stability. Rapsyn may function as a scaffolding
protein
presenting a MuSK-activated Src-related kinase to the
AChRs [43].

Upon phosphorylation and activation,
MuSK interacts with a large number of effectors and pathways converging
toward the regulation
of AChR clusterization through different mechanisms.
MuSK indeed controls (1) AChR scaffolding and redistribution via actin
cytoskeleton reorganization, (2) AChR stabilization
through AChR phosphorylation, (3) its proper endocytosis and turnover
are also important for AChR clustering, and (4)
enhancement of transcription of AChR genes selectively in subsynaptic
nuclei
(reviewed in [14]).

AChR subunits are selectively
transcribed in the synaptic nuclei of myofibers. mRNAs encoding other
synaptic proteins such
as AChE, MuSK, and rapsyn are also concentrated at the
synapses. The pathway activating AChR transcription in the subsynaptic
regions still remains unclear. One candidate activator
is neuregulin-1 (NGR-1), a glycoprotein which works as an extracellular
signal that stimulates AChR and other synapse-specific
components transcription. NRG-1 acts by binding to membrane-associated
tyrosine kinase receptors related to the EGF receptor
(erbB proteins 2-3-4) [44] (Fig. 1b). In vivo studies on NGR-1 role in NMJ differentiation are difficult because nrg-1−/−embryos die far before NMJs start forming. Heterozygous mice are viable and fertile and show a decreased number of AChRs at
the NMJs [45, 46]. However, some recent in vivo studies, in which neuregulin-1 and erbB proteins were conditionally deleted, demonstrated
that neuregulin signaling is dispensable [47, 48].
For this reason, the idea that NRG-1 is the main signal responsible for
the increased transcription of AChRs is highly
controversial. Recent evidences also indicate that
neuregulin may have a role in AChR clustering and trafficking by
modulating
rather than determining AChR expression at the NMJs [49].

The pathways leading from NRG-1–ErbB binding to regulation of AChR gene transcription have been identified. Both in cultured
muscle cells and in vivo, ErbB phosphorylation by NRG-1 concomitantly stimulates ERK [50, 51] and the c-JUN N-terminal kinase (JNK) activity [52, 53].
The activation of these pathways results in increased levels and
phosphorylation of the E-twenty-six (ETS)-domain-binding
transcription factor GA-binding protein (GABP)-α which
binds DNA, heterodimerizes with GABP-β, and increases AChR
transcription
[54, 55] (Fig. 1b). ETS transcription factors bind DNA at the N-box, a DNA element located not only on the AChR promoter but also on the promotor
of other NMJ components (utrophin and acelylcholinesterase) [56–58]. In vivo inhibition of GABP results in reduced expression of AChRs [59]
indicating the GABP signaling pathway, activated directly or indirectly
by neuregulin, is necessary for the formation of
functional synapses. As stated before, the agrin–MuSK
pathway also contributes to increase synaptic AChR gene transcription
by a NRG/ErbB-independent but Rac-dependent JNK
activation (Fig. 1b) and also by interacting with the NRG/ErbB pathway [43, 60, 61].

3.2 Negative signals

The positive signals we described
enhance AChR accumulation only in the synaptic area. To achieve a
correct formation of AChR
clusters in the NMJs, the presence of some negative
signals that decrease AChR concentration in the rest of the fiber, in
non-synaptic areas, is necessary.

Muscle depolarization induced by released ACh is a negative signal for AChRs. It inhibits AChR localization, stability [62], and transcription along the muscle fiber by stimulation of cyclin-dependent kinase 5 [63, 64], protein kinase C (PKC), and Ca2+/calmodulin-dependent kinase II (CaMKII) [65]. Indeed, mutant mice lacking acetyltransferase, a biosynthetic enzyme for ACh, develop faster and larger AChR clusters;
they also exhibit hyperinnervation due to a broader distribution of AChR clusters along the muscle fiber [66, 67].

In detail, through muscle action
potential, ACh induces downregulation of AChR transcription along the
entire fiber. However,
since the previously described positive signals
enhance AChR accumulation only in the synaptic area (Fig. 1), this results in a ACh-dependent suppression of AChR subunit gene transcription in extrasynaptic nuclei (Fig. 2) [68].
Suppression of AChR transcription occurs through myogenin inhibition.
Myogenin is a basic helix-loop-helix myogenic transcription
factor that activates AChR genes in the absence of
innervation or by blockade of muscle electrical activity by denervation
[69]. Myogenin
regulates AChR and many other muscle gene transcription by binding the
E-boxes located on their promoter/enhancers.
Myogenin is downregulated as the muscle matures
around the time when innervation is completed, and it is upregulated
upon
denervation [70]. Forced expression of myogenin in vivo is able to induce AChR expression along the entire muscle fiber [71].
Multiple mechanisms have been proposed to explain how neural activity
controls myogenin in postnatal muscle. On the basis
of one of these mechanisms, following muscle
depolarization, extracellular calcium enters through voltage-activated
channels
and activates different signal transduction
cascades mediated by PKC and CaMKII, leading to myogenin phosphorylation
[63, 72].
This posttranslational modification inhibits myogenin binding to
E-boxes of AChR promoters, resulting in reduced AChR expression
(Fig. 2) [73, 74]. A second regulatory mechanism restraining myogenin activity involves transcriptional repression of myogenin (Fig. 2). The posttranslational and the transcriptional mechanisms of myogenin repression could act on a different timescale to ensure
that myogenin expression remains blocked as long as the muscle is innervated [30].
Although it is very well known that myogenin is transcriptionally
modulated as a consequence of muscle depolarization,
the mechanisms that are upstream of this regulation
have only recently started to be elucidated. Several studies reported
that in myogenin repression during innervation,
class II deacetylases are recruited at the myogenin promoter. The
hystone
deacetylase 9 (HDAC9) splice variant MITR is a
transcriptional repressor induced in innervated muscle. It contributes
to myogenin
repression by chromatin acetylation and acts as a
corepressor in a complex with the myocyte enhancer factor-2 (MEF2)
transcription
factor (Fig. 2) [75]. Another myogenin repressor is dachshund homolog 2 (DACH2) (Fig. 2). It has been shown that, during denervation, DACH2 is inhibited by HDAC4, another class II deacetylase, which induces myogenin
expression thereby activating AChRs [76–78].
A third circuit of postnatal myogenin expression regulation is mediated
by MSY3/CsdA, a Y-box factor that binds the myogenin
promoter at a new binding motif (highly conserved
element, HCE) and exerts a repression function (Fig. 2). MSY-3 is therefore partially responsible for the restricted spatiotemporal expression of AChR in the subsynaptical regions
of muscle fibers [79]. The conserved binding motifs of HDAC9/MEF2, DACH2, and MSY-3 are adjacent sequences on the myogenin promoter, which suggests
a strong interplay between the three cis–trans systems to ensure a proper myogenin and AChR repression in adult and innervated muscle.

Fig. 2 The
ACh–AChR interaction triggers NMJ assembly negative signals. Global
AChR transcription is inhibited by ACh released by
the nerve. AChR gene transcription suppression
occurs through myogenin inhibition. The myogenic transcription factor
myogenin
activates muscle-specific genes including AChRs
by binding the E-box element. When the action potential reaches the axon
terminals,
it induces the opening of the voltage-gated Ca2+ channels on the presynaptic nerve membrane. The Ca2+
influx into the neuron leads synaptic vesicles to fuse with the
presynaptic membrane and to release the neurotransmitter
acetylcholine (ACh) in the synaptic cleft. From
here, ACh diffuses and binds to acetylcoline receptors (AChRs) localized
on
the postsynaptic membrane on the muscle fiber.
This binding makes AChRs permeable to ions so that Na+ flows into the fiber while K+ flows out of the muscle cytosol. In this way, the muscle membrane locally depolarizes, and this local depolarization opens
voltage-gated Na+ channels on the membrane, allowing a propagation of the depolarization (action potential) that spreads to involve the entire
plasma membrane. The action potential induced by ACh release leads to the release of Ca2+ from the sarcoplasmic reticulum into the cytosol which causes muscle contraction. The high concentration of Ca2+
inside the cytosol also activates CaMKII and PKC kinases which mediate
signaling pathways leading to myogenin phosphorylation
and inactivation. When myogenin is inactive,
AChR expression is suppressed. In addition to myogenin phosphorylation, a
second
mechanism, which represses myogenin, is
activated by Ca2+ signaling upon innervation and leads to the
activation of three transcription repressors (MSY-3, DACH2, and HDAC9).
These
repressors bind myogenin promoter and inhibit
its transcriptional activation. The binding motifs for MSY-3, DACH2, and
HDAC9
(respectively HCE, SIX, and MEF2) are located on
adjacent sequences on the myogenin promoter

In conclusion, while during development
the first events leading to AChR clusterization are MuSK- and
rapsyn-dependent but
nerve-independent (AChR prepatterning), the
stabilization of some prepatterned AChR clusters requires the
innervation. Indeed,
once the muscle is contacted by the nerve, ACh
released by the motor neuron induces a postsynaptic potential which
stabilizes
previous AChR clusters in the contacted area and
prevents AChR clustering in non-contacted area. Moreover, agrin released
by the neuron also stabilizes the AChR clusters and,
as well as neuregulin, strongly increases AChR transcription in
subsynaptic
nuclei (Fig. 1). Concomitantly, Ca2+-dependent signals triggered by nerve-induced muscle electrical activity cause myogenin downregulation and inhibition of AChR
transcription in extrasynaptic regions of the muscle fiber (Fig. 2). The integration of these two mechanisms makes possible the compartmentalization of AChR gene expression in subsynaptic
nuclei and the stabilization of AChR clusters only at NMJs.

4 Neuromuscular disorders associated to defects in synaptogenesis

When NMJs are severely impaired, the
muscles develop congenital myasthenic syndromes. In such disorders,
defects in neuromuscular
transmission cause a fatigable weakness in limb, ocular,
bulbar, truncal, and respiratory muscles. Generally, weakness becomes
more severe with exercise and improves with rest. CMS
have been identified worldwide and occur at a frequency of <1/500,000
people, although this incidence is constantly growing [80].
Diagnosis can be difficult, often requiring a high risk of clinical
suspicion. Most patients present symptoms during infancy,
although in some syndromes, symptoms are not manifested
until childhood or adult life. Depending on the neuromuscular
transmission
defect, CMS can be classified as presynaptic, synaptic,
or postsynaptic, even though it is not always possible to identify
accurately the cause of the pathology [81].
Over the past few years, many causative genes and mutations for CMS
have been identified, although in many patients, no
causative mutations can be found. In most cases, CMS
causative mutations are missense, truncation, or splice-site mutations
and result in structural changes of AChRs or in AChR low
affinity for ACh. CMS-associated genetic mutations have been mapped
in AChR subunits, choline acetyltransferase, the collagen
tail subunit of acetylcholinesterase, rapsyn, MuSK, and skeletal
muscle–sodium channel Na1.4 [82–84].

Dok-7 CMS is a newly identified synaptopathy characterized by a “limb girdle” phenotype that mainly affects proximal muscles
rather than the distal ones with ptosis present from early age [85].
Mutations in the Dok-7 locus were identified in groups of patients who
had small EPs, a reduced number of AChRs at the
EPs, and a limb-girdle phenotype with no mutations in
rapsyn or AChRs. Dok-7 mutants generally have abnormally small and
simplified
NMJs but show normal AChR and AChE functions, correct
MuSK activation, and AChR clusterization, although Dok-7 acts in concert
with MuSK in activating rapsyn to concentrate AChRs at
the junctional folds. It has therefore been suggested that the altered
size and integrity of NMJs observed were probably the
consequence of a high and widespread degeneration and remodeling of
the EPs [86, 87].

Together with genetic alterations in the
coding region of components of the synaptic apparatus, also mutations in
their regulative
regions can account for the congenital myasthenic
phenotype. A mutation in the N-box of the AChRε subunit promoter,
resulting
in a decreased GABP transcription factor binding activity
[88], causes a reduction in the AChR number which is the cause of CMS clinical symptoms in human [89, 90].

Besides being congenital, myasthenia can
also be caused by a complex autoimmune disorder of neuromuscular
transmission (myasthenia
gravis, MG). MG is characterized by a fluctuating
weakness of the head, neck, and upper extremity muscles that worsens
with
activity and improves with rest [91, 92].
When the bulbar and the respiratory muscle deteriorate, the disease
becomes life-threatening, and ventilation with medication
is required. Approximately 80% of MG patients have
autoantibodies against AChR and, consequently, they show a decreased
number
of AChRs at the postynaptic membrane [93].
In addition, the deposit of the complement causes a structural and
functional damage of the postsynaptic membrane, resulting
in a neuromuscular transmission failure. In some MG
patients, autoantibodies against MuSK have also been found, but their
pathogenic role remains unclear since the number of AChRs
is not reduced and no deposits of complement are found at the
postsynaptic
membrane. It has been therefore suggested that, since
MuSK can be required for retrograde signal, it is possible that MuSK
autoantibodies interfere with the presynaptic apparatus
organization [94, 95]. Some other MG patients have autoantibodies to Lrp4, which inhibit Lrp4 function [96].

5 NMJ degeneration in sarcopenia

The age-related muscle loss (sarcopenia)
strongly impairs the quality of life in the elderly, and it is
associated with increased
risk of morbidity and mortality [97].
The etiology of sarcopenia is not clear yet. However, many studies
suggest that NMJ structural and functional impairment
plays a key role in muscle wasting during aging. NMJ
degeneration is a feature of sarcopenia, and it has been documented both
in animal models and in humans. Changes in NMJ morphology
depend on the type of muscle and include variation of the nerve
terminal area size, of the end plate size, of the number
of synaptic vesicles, of mitochondrial content, and of smooth
endoplasmic
reticulum content (reviewed in [98]).
By in vivo live animal imaging, it has been revealed that at
12–18 months, in mice, a few NMJs start to display loss of
motor neuron terminal branches and AChRs. At this point,
probably in order to compensate for loss of some synaptic sites,
motor neuron sprouting increases and accounts for the
formation of new NMJs. However, this compensatory effect strongly
decreases
after 18 months of age. Moreover, the newly formed
synapses are more sensitive to degenerative changes. Therefore, by
24–36 months,
most NMJs are completely denervated, and postsynaptic EPs
are highly fragmented [98].

Many mechanisms have been evoked as cause
of NMJ degeneration during aging. Age-related changes in the morphology
of NMJs
can be related to alteration of the axonal transport,
which impairs the availability of trophic factors and organelles, such
as mitochondria, essential for neuronal survival [99]. Age-related changes in myelinated Schwann cells have also been reported and might be associated with NMJ degeneration [100].
It has interestingly been shown that neurotrophic and myotrophic
factors such as BDNF, NT-3, insulin-like growth factor
(IGF)-I, and IGF-II, which are necessary for the
maintenance of presynaptic and postsynaptic apparatus at the NMJ, also
play
a modulating role in aging [101, 102]. For example, IGF-1 injections into muscle inhibit motor neuron and NMJ degeneration and prevent age-related force decline
in mice [103].

Recent studies suggest that motor neuron degeneration and consequent denervation of myofibers are a major cause of muscle
mass loss [104, 105].
In aged people, muscle fibers undergo cycles of denervation and
innervation. During these cycles, some myofibers are lost,
and others, which were innervated by fast motor neurons
(type II fibers), are reinnervated by slow ones (type I fibers). This
results in an increased percentage of type I fibers and
atrophy, which characterizes sarcopenia. It has also been demonstrated
that chronic exercise training and higher neuromuscular
activity counteract the alterations in motor neurons and delay the
onset of denervation and sarcopenia in aged people [104–106].

Although the causes of age-associated NMJ
degeneration remain unclear, other mechanisms might contribute to
compromised muscle
neurotransmission and sarcopenia in aging. For example,
sarcopenia is also characterized by mitochondrial dysfunctions.
Presynaptic
terminals and postsynaptic EPs contain a high
concentration of mitochondria, which are critical for NMJ function since
they
account for energy support, Ca2+ buffering, synaptic transmission, and apoptosis [107].
Mitochondrial dysfunctions might lead to altered calcium buffering,
less ATP generation and more ROS production, and oxidative
damage during aging, which impairs NMJs [108, 109]. In particular, the impaired buffering of Ca2+ determines an increased concentration of cytosolic Ca2+,
which might activate some proteases called calpains. It has been shown
that calpains interact with rapsyn thereby disrupting
AChR clusters. Through this mechanism, mitochondrial
impairment might partially account for the age-related dispersion of
AChR clusters [110].
Moreover, a direct correlation between increased oxidative stress and
neuromuscular function has been recently proposed.
Mice lacking the antioxidant enzyme CuZnSOD (Cu/Zn
superoxide dismutase, Sod 1) show high oxidative damage and rapid
sarcopenia.
The morphology of NMJs at 11 months in Sod1−/−mice is altered, and it is comparable to NMJ degeneration in 33-month-old, wild-type mice. Furthermore, denervated NMJs and
fragmentated AChRs observed in young Sod1−/−mice notably reduce muscle contractile force [111, 112].
These observations suggest that NMJ degeneration during aging might be
caused also by age-related oxidative stress, which
usually is produced by mitochondrial dysfunctions.
Oxidative stress also induces damage in myelinated peripheral nerves and
consequent increased inflammatory cytokines during aging
(Reviewed in [98]).

Interestingly, it has been recently reported that calorie restriction slows down the aging process and, in particular, decreases
deterioration of peripheral nerve by upregulating autophagy [113]. It has also been shown that calorie restriction reduces oxidative damage, NMJ degeneration, and muscle atrophy in Sod1−/−mice ,and this further suggests that oxidative damage affects NMJs [111, 114].

6 Conclusions and prospects

The complexity of the neuromuscular
junction apparatus and of the regulative systems responsible for its
organization indicates
that the functional modulation of the connection between
neurons and muscle fibers is crucial. The right concentration of
the acetylcholine receptors in the area where the axon
terminals juxtaposes the postsynaptic membrane accounts for the NMJ’s
ability to generate the action potential that provokes
muscle contraction and consequently movements. For this reason, multiple
levels of regulation are committed to accurately control
this process. Animal models show that impairing only one of these
regulative pathways can cause severe phenotypes
characterized by compromised coordinate movements and breath capacity
which
usually prove fatal. We have also seen that
transcriptional control plays an important role in muscle physiological
response
to nerve activity. CMS and myasthenia gravis are human
pathologies characterized by an extreme fatigability and muscle weakness
and in which insufficiency of a single component of NMJ
frequently accounts for their pathophysiology. Studying NMJ transmission
in aging and in those pathological conditions
characterized by different levels of muscle weakness may reveal new
mechanisms
that contribute to the fine-tuning of the interplay
between motor neurons and muscles. Understanding the mechanisms
underlying
age- and disease-associated atrophy and alteration of
NMJs should open up new potential therapeutic avenues to these skeletal
muscle dysfunctions.

Acknowledgments

The
authors have been suppported in this work by the European Union Grant
SICA-HF project. All authors of this manuscript
comply with the guidelines of ethical authorship and
publishing in the Journal of Cachexia, Sarcopenia and Muscle [115]. European Union Seventh Framework Program FP7/2007-2011 under grant agreement 241558 (SICA-HF).

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